Rapid Optogenetic Clustering in the Cytoplasm with BcLOVclust.

Area of Science:

• Optogenetics and synthetic biology
• Molecular and cellular biology
• Biochemistry

Background:

• Optogenetic control using protein clustering is powerful but limited by the scarcity of suitable light-inducible oligomerizing proteins.
• The photoreceptor BcLOV4 induces protein clustering with blue light, but its membrane association restricts its application.
• Existing optogenetic tools like Cry2 have limitations in clustering kinetics and multiplexing.

Purpose of the Study:

• To engineer a BcLOV4 variant that clusters in the cytoplasm, independent of membrane binding, for enhanced optogenetic applications.
• To characterize the clustering and de-clustering kinetics of the engineered variant.
• To explore strategies for modulating clustering magnitude and enabling multiplexed optogenetic control.

Main Methods:

• Site-directed mutagenesis of BcLOV4 to identify key residues coupling clustering to membrane binding.
• Engineering and characterization of the cytoplasmic clustering variant, BcLOVclust.
• Kinetic analysis of clustering and de-clustering, optimization of clustering magnitude, and assessment of temperature sensitivity.
• Demonstration of multiplexing with Cry2 and application in controlling signaling proteins and stress granules.

Main Results:

• Identification of key amino acids enabling BcLOVclust to cluster in the cytoplasm without membrane association.
• BcLOVclust exhibits significantly faster clustering and de-clustering kinetics compared to Cry2.
• Clustering magnitude can be enhanced by fusing with intrinsically disordered regions (e.g., FUS) or specific fluorescent proteins.
• BcLOVclust activity is temperature-sensitive, with faster cluster dissolution at higher temperatures.
• Successful multiplexing of BcLOVclust and Cry2 in the same cells at low temperatures.
• Demonstrated utility of BcLOVclust for controlling signaling proteins and stress granules.

Conclusions:

• BcLOVclust represents a novel, rapidly cycling optogenetic clustering module functional in the cytoplasm.
• The engineered variant overcomes limitations of previous BcLOV4 applications and offers advantages over Cry2 in kinetics.
• While currently best suited for sub-30°C applications, further understanding of its thermal response could expand its use to physiological mammalian temperatures.